Checkerboard Method

The checkerboard method was used to determine the potency of the combination of PA with CLR or MTZ on H. pylori. The assay assesses the activities of antimicrobial combinations tested at concentrations in serial two-fold dilutions under MIC. Autoclaved MH agar (5% sheep blood) was prepared for gradient dilutions of PA, CLR, and MTZ. The final concentration of each drug ranges from 1 to 0.25 MIC; the MIC values are shown in Table 1. And according to the checkerboard method, to design different combinations of the two drugs, their combined effects must be observed. H. pylori was then resuspended in PBS, and the concentration was adjusted to McFarland 1 (1 × 10⁸ CFU/ml) through turbidimetry. Bacterial suspension (100 μl) was spread onto the drug-containing MH agar plates and cultured in a tri-gas incubator for 4 days. The fractional inhibitory concentration index (FICI) was calculated as the sum of the MIC of each compound used in combination and divided by the MIC of each compound used alone as follows:

MICs for H. pylori^a.

The results were interpreted using the following criteria: synergism (FICI ≤ 0.5), additive (0.5 < FICI ≤ 1), indifference (1 < FICI < 4), and antagonism (FICI ≥ 4) (Krzyzek et al., 2020).